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Received: 17 December 2020 Accepted: 18 November 2022 Published online: 19 January 2023

DOI: 10.1002/agj2.21264

ORIGINAL ARTICLE
A g ro n o m y, S o i l s , a n d E nv i ro n m e n t a l Q u a l i t y

Impacts of long-term fertilization on crop yield and microbial

communities under rice–rapeseed rotation

Jifu Li Guoyu Gan Qianqian Zeng

Engineering Research Center of Ecology

and Agricultural Use of Wetland, Ministry
of Education/College of Agriculture,
Yangtze University, Jingzhou, China
Correspondence
Li Jifu, No. 266, Jingmi Road, Jingzhou
District, Jingzhou City 434023, Hubei
Province, China.
Email: jifuli@yangtzeu.edu.cn
Assigned to Associate Editor Xinda Lu.
Funding information
Engineering Research Center of Ecology
and Agricultural Use of Wetland, Ministry
of Education, Grant/Award Number:
KF2020015; Key Laboratory of Fertilizer
Utilization, Ministry of Agriculture and
Rural Affairs, China, Grant/Award Number:
KLFAW201901
Abstract
Soil microbes play an integral role in agricultural production and soil fertility, while
limited information is available about the effects of long-term fertilization on rice
(Oryza sativa L.)–rapeseed (Brassica napus L.) rotation yield, soil properties, and
microbial communities in central China. Here, we report the response of plant–soil
system to different mineral fertilizer inputs, including the balanced fertilization with
N, P, and K (NPK) fertilizers and unbalanced fertilizers without one of the major
nutrients (PK, NK, and NP) for 10 years. Compared with the NPK treatment, the
average annual decrease rates of rice and rapeseed yield in the PK, NK, and NP
treatments were 12.5%, 18.1%, 6.4% and 33.1%, 54.8%, 16.0%, respectively. Long-
term fertilization significantly changed the soil chemical properties, especially the
pH and available K content. Compared with the NPK fertilization, the NK treatment
increased the soil microbial composition and diversity, whereas PK and NP treat-
ments had a reverse impact. Available K, pH, and slowly available K were closely
related to alterations of the dominant bacterial community. On the contrary, the
Olsen-P, slowly available K, and pH were significantly correlated with the fungal
community. Soil properties changed by fertilization had a more direct effect on crop
yield than the microbial community; moreover, the bacterial group had a higher influ-
ence than the fungal group on crop yield. Therefore, a balanced nutrient input using
NPK fertilization is an effective management strategy for soil biological fertility and
sustainable crop yield.

1 INTRODUCTION example, soil acidification, nutrient loss, and land capacity

Mineral fertilizer, especially nitrogen (N), phosphorus (P),
and potassium (K), is a large contributor to global food pro-
duction. Reasonable fertilization can promote the positive
development of the soil ecosystem; however, inappropriate
or excessive fertilization will cause production reduction and
a series of environmental problems in agro-ecosystem, for
Abbreviations: ACE, abundance-based coverage estimator; GOF,
goodness of fit; OTU, operational taxonomic units; SOM, soil organic
matter.
degeneration (Lu et al., 2018; Reed, 2017; Zhang et al., 2020).
As a vital component of the soil ecosystem, microorganisms
play a critical role in the formation of soil fertility and the bio-
geochemical cycle of nutrients (Ullah et al., 2020). The effect
of fertilization on soil microorganisms is different due to the
quantity, type, time of fertilization, and the combination of
different types of fertilizers. Meanwhile, soil microorganisms
respond to different fertilizer rates, leading to changes in the
biomass, activity, and community structure (Ling et al., 2017;
Wang et al., 2018; Wasaki et al., 2018).

© 2022 The Authors. Agronomy Journal © 2022 American Society of Agronomy.

208 wileyonlinelibrary.com/journal/agj2 Agronomy Journal. 2023;115:208–221.


LI ET AL.
Previous studies have shown that fertilization could alter
the nutrient content of soil (i.e., total N, available P, and
available K) and directly drive the evolution of soil microbial
communities (Cookson et al., 2005; Mooshammer et al.,
2014). However, other studies believe that fertilization indi-
rectly changes soil properties. Acting on soil microorganisms,
Zeng et al. (2016) found that the decrease in soil pH caused
by the application of N fertilizer was the primary cause of
bacterial community changes. Long-term P fertilizer input
increased soil microbial P immobilization by decreasing the
relative abundance of the P-starvation response gene (phoR)
and increasing the relative abundance of the low-affinity
inorganic phosphate transporter gene (Dai et al., 2020). In
black soil, Proteobacteria, Acidobacteria, and Actinobacteria
were the dominant bacteria when different amounts of K
fertilizer were applied, while the relative abundance of Aci-
dobacteria was significantly decreased with the increase of
K application (Yu et al., 2019). Fan et al. (2018) showed that
some keystone species of bacteria had higher functional gene
copies associated with N and P element cycling in controlling
soil function and crop production. Meanwhile, the microbial
community was highly responsive to K fertilization, which
was associated with lower upland crop yield and higher
abundance of potential fungal pathogens. However, the indi-
vidual effects of N, P, and K (NPK) fertilizers and balanced
fertilizer inputs on bacterial and fungal communities, and
the potential effects of fertilization-induced microbial groups
on rotation system productivity in paddy soils have not been
addressed.
Rice (Oryza sativa L.)-based cropping system is the
most widely distributed and productive agroecosystems in
Southern China, Southeast Asia, and South Asia. The joint
contribution rate of NPK fertilizers application to crop yield
was more than 50% in China (Tian et al., 2018). Eo and Park
(2016) illustrated that the application of N fertilizer or P fer-
tilizer alone reduced the bacterial diversity and simplified
the interactions among dominant bacterial members. With
the project on soil testing for formulated fertilization in a
pilot area carried out in 2005, balanced fertilization has been
widely accepted by farmers, but insufficient or excessive fer-
tilization also took place in rice region of the Yangtze River
Basin. There is not enough information on the effects of indi-
vidual nutrient input deficiency stress (N, P or K fertilizer) and
balanced fertilization on soil biological fertility; especially,
there is a lack of long-term field comparative studies on paddy
soil (Radhakrishnan & Varadharajan, 2016). Therefore, we
carried out this experiment to assess the relationships between
soil properties, microbial community altered by fertilization
with crop yield.
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209
Core Ideas
∙ Crop yields were sensitive to P deficiency than N
and K deficiencies.
∙ Coupling interaction existed between soil proper-
ties and microbes on yield.
∙ The responses of microbial diversity to P, K, and N
deficiency were different.
∙ Available K, pH, and slowly available K were
closely related to bacterial community.
∙ Olsen-P, slowly available K, and pH were signifi-
cantly correlated with fungal community.
2 MATERIALS AND METHODS
2.1 Experimental site description
A long-term field trial was initiated in 2011 at the Shuangdian
Experimental Station located in Jingzhou City, Jianghan Plain
(N 30˚33′24″, E 112˚4′56″, altitude 52 m), Hubei Province,
central China, adopting a middle rice–rapeseed (Brassica
napus L.) rotation system developed in 1999 (Tang et al.,
2018). Monthly temperature and rainfall during the exper-
iment (2011–2020) are shown in Figure S1. The average
annual rainfall was 1140 mm, and the air temperature was
15˚C. Soil type was classified as Alfisol using the Word Soil
Classification of the Food and Agriculture Organization (sand
26.3%, silt 40.8%, and clay 32.5%). At the beginning of the
experiment (June 2011), the selected soil basial properties at a
depth of 0–20 cm were as follows: pH 6.4, soil organic matter
(SOM) 28.8 g kg−1 , total N 0.6 g kg−1 , available N 275.3 mg
kg−1 , Olsen-P 4.44 mg kg−1 , available K 96.6 mg kg−1 , and
slowly available K 528.2 mg kg−1 . According to the nutrient
deficiency index of paddy fields, the available nitrogen con-
tent for the field experiment is high, indicating that the field
soil is rich in N and K but deficient in P (Tang et al., 2018).
2.2 Experiment design
A complete randomized block design was conducted with four
treatments and three replications. The treatments were as fol-
lows: (1) NPK: balanced fertilizers of NPK were applied; (2)
PK: mineral fertilizers of P and K were applied; (3) NK: min-
eral fertilizers of N and K were applied; and (4) NP: mineral

210
fertilizer of N and P were applied. The regional balanced fer-
tilizer application for annual rice and rapeseed season were
180 kg N ha−1 , 90 kg P2 O5 ha−1 , 120 kg K2 O ha−1 , and
1.6 kg B ha−1 only for rapeseed season (Yousaf et al., 2017).
During rice season, N (urea, 46% N) was applied in three
splits: 60% as basal fertilizer before rice transplanting, 20%
at tillering stage, and 20% at the booting stage. K (potassium
chloride, 60% K2 O) was applied as 60% basal fertilizer and
40% booting fertilizer. P (superphosphate, 12% P2 O5 ) was
applied manually as basal fertilizers. During oilseed rape sea-
son, N was applied in three splits: 60% as basal fertilizer, 20%
during the overwintering stage, and 20% at the beginning of
stem elongation. P, K, and B (sodium borate, 11% B) fertiliz-
ers were applied manually as basal fertilizers. After each crop
was harvested, the crop straw was removed from the plots.
The specific fertilization time for a crop is shown in Figure
S1. Local crop varieties, Eke No. 1 and Huashuang No. 11,
widely planted in the experimental region with high yields
and extensive adaptability, were used for rice and rapeseed,
respectively. The dimensions of the plot were 20 m2 , with a
length of 5.0 m and a width of 4.0 m.
2.3 Methods
2.3.1 Crop harvest
For each season, the whole plots of mature rapeseed and rice
plants were harvested and thrashed in each plot. The grains
were dried to determine the grain yield.
2.3.2 Soil sampling and preparation
On September 10, 2020 (1 day before rice harvest), soil sam-
ples were randomly collected from four points in each plot
between the rice lines at 0–20 cm depths by using an auger
with a diameter of 5.0 cm. Soil from the four core samples of a
plot was mixed to obtain one composite sample. After remov-
ing stones, roots, and plant residue using a 2-mm mesh, each
sample was divided in half: one half was air-dried for physico-
chemical analyses, and the other half was immediately stored
at −80˚C for DNA extraction and microbiological analysis.
2.3.3 Determination of soil physicochemical
indexes
The air-dried soil samples were used to determine physico-
chemical properties according to the methods described by
Ding et al. (2016). Briefly, soil pH was measured in water
(1:2.5 w/v) by a pH meter (PHS-3C, INESA Scientific Instru-
ment, Shanghai, China). Olsen-P was extracted with 50 ml
of 0.5 mol L−1 NaHCO3 (pH 8.5) and determined using an
injection pump analyzer (AA3, Bran+ Luebbe, Germany).
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LI ET AL.
Available K and slowly available K were extracted with 1 mol
L−1 NH4 OAc and 1 mol L−1 HNO3 solution, respectively, and
measured by a photoelectric flame photometer. The SOM was
determined using a wet oxidation procedure with potassium
dichromate (K2 Cr2 O7 )-sulfuric acid (H2 SO4 ). Furthermore,
soil available N was determined by the alkaline hydrolysis
diffusion method.
2.3.4 Soil DNA extraction and
high-throughput sequencing analysis
DNA was extracted from the soil samples (0.5 g) using a
Fast DNA Spin Kit for Soil (MP Biomedicals, USA) in accor-
dance with the protocol of the manufacturer. The quantity and
quality of the DNA extracts were determined using a Nan-
oDrop 2000 spectrophotometer (Thermo Fisher Scientific).
The extracted DNA was stored at −20˚C for further analyses.
An aliquot of the extracted DNA from each sample
was used as the template for amplification. The V3–V4
hypervariable regions of the bacterial 16S rRNA gene
sequences and the ITS region of the fungal rRNA gene
sequences were amplified (Wang et al., 2017). Amplicon
libraries were prepared using tagged bacterial and fun-
gal universal primers, that is, 338F and 806R for bacte-
ria and ITS1F and ITS2R for fungi. The DNA samples
were amplified individually using the fusion primer pairs
338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R
(5′-GGACTACHVGGGTWTCTAAT-3′) for bacteria and
ITS1F (5′-CTTGGTCATTTAGGAAGTAA-3′) and ITS2R
(5′-GCTGCGTTCTTCATCGATGC-3′) for fungi to generate
polymerase chain reaction (PCR) fragments. The following
thermal program was used for amplification: initial denatura-
tion 98˚C for 2 min, followed by 27 cycles of denaturation at
98˚C for 15 s, annealing at 55˚C for 30 s, extension at 72˚C
for 30 s, and a final extension at 72˚C for 5 min. The PCR
reactions were performed in a 25 μl mixture containing 5 μl
of 5× reaction buffer, 5 μl of 5×GC buffer, 2 μl of dNTP
(2.5 mM), 1 μl of forward primer (10 μM), 1 μl of reverse
primer (10 μM), 2 μl of DNA template, 8.75 μl of ddH2 O, and
0.25 μl of Q5 DNA polymerase (Tian et al., 2017). The PCR
products were purified using the AxyPrep DNA Gel Extrac-
tion Kit (Axygen Biosciences) and quantified using Quantus
Fluorometer (Promega). The target sequences were performed
on an Illumina MiSeq 250 sequencing platform by Shanghai
Personal Biotechnology.
2.3.5 Sequence processing
In sequencing the original data to remove the primer adapter
sequence, the processed low-quality bases were removed,
and then the remaining data were spliced to obtain valid
sequence data for each sample. Finally, using 97% as the

LI ET AL.
threshold, the 16S and ITS sequences were divided into oper-
ational taxonomic units (OTUs). Using QIIME 2 software, the
UCLUST sequence comparison tool was used to cluster with
97% sequence similarity and each sequence with the highest
OTU degree was selected as the representative sequence of
the OTU (Caporaso et al., 2010). For bacterial 16S rRNA and
fungal ITS genes, both the Greengenes database and the Silva
database were used as template sequences for OTU classifica-
tion status identification (Edgar, 2010). After quality filtering
and removal of chimeric sequences, 364,587 and 336,310
high-quality sequences were, respectively, clustered into
48,847 and 76,607 OTUs for each bacterial and fungal sample.
2.4 Statistical analysis
The analysis of variance procedure in SPSS 16.0 (SPSS
Inc.) was used to perform data analysis on soil biogeo-
chemical properties and alpha diversity. Differences at the
p < 0.05 level was considered statistically significant, as deter-
mined using the least significant difference test. The Chao1,
abundance-based coverage estimator (ACE), and Shannon,
Simpson indexes were used to estimate the alpha diversity of
each sample using MOTHUR (Schloss et al., 2009). To deter-
mine the structural differences between bacterial and fungal
communities at different treatments, an analysis of similarities
was also conducted using QIIME2 based on Bray–Curtis dis-
tance measurements and abundance data (Wang et al., 2017).
To determine which taxa were significantly affected, the linear
discriminant analysis effect size algorithm was implemented
(Segata et al., 2011). The “vegan” package in R 4.1.0 language
was used to perform similarity analysis, and redundancy
analysis (RDA) was used to access the effects of soil environ-
mental factors on bacterial and fungal communities (Oksanen
et al., 2010). To reveal the potential pathways (soil proper-
ties, alpha diversity, and microbial community) that influence
rice yield and rapeseed yield, partial least squares path models
were evaluated using the goodness of fit (GOF) statistic (Bar-
beran et al., 2014) and assembled by the “inner plot” function
using the “plspm” package of R 4.1.0.
3 RESULTS
3.1 Crop yield
Over the 10 years, crop yields were significantly affected
by different fertilization for rice and rapeseed (Figure 1).
The grain yields with NPK fertilizer were 8591 kg ha−1
and 2325 kg ha−1 annual average for rice and rapeseed,
respectively. Compared with the NPK treatment, the aver-
age annual decrease rates of rice and rapeseed in the PK,
NK, and NP treatments were 12.5%, 18.1%, 6.4% and 33.1%,
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211
54.8%, 16.0%, respectively. In the first three crop rotations,
the rice yield from the PK, NK, and NP treatments did not
show a significant reduction, whereas after the four rotation
cycles, a significant decreasing tendency could be seen in
Figure 1a and Figure S2. The rice yield followed the order of
NPK > NP > PK > NK (Figure 1b). For the subsequent crop
rapeseed, a yield decrease effect appeared in the second rota-
tion, especially in the NK treatment and PK treatment, which
are shown in Figure 1c and Figure S2.
3.2 Soil properties
The results in Table 1 showed that among the four treatments,
there were significant changes in the soil chemical properties.
Compared with the initial value, the pH for the NK, NP, and
NPK treatments had a downward trend. The PK fertilization
had no significant difference from the initial value. Compared
with the initial value, the SOM content of each treatment
increased to varying degrees, and the rate of increase in the
NPK fertilization was the highest, reaching 16.3%. The results
of available N content showed that the content of the PK,
NK, and NP treatments showed a downward trend, decreas-
ing by 12.4%, 4.7%, and 5.7%, respectively, while that of
the NPK fertilization increased by 4.0%. Compared with the
initial value, the NK treatment significantly reduced the soil
Olsen-P content by 18.0%, and there was no significant differ-
ence in other treatments. Ten years of NP fertilization led to
a decrease in the available K content and the slowly available
K content in soil, which were 25.2% and 4.9% lower than the
initial values, respectively. In comparison, the NPK treatment
increased the available K and the slowly available K contents
by 14.7% and 1.4%, respectively.
3.3 Alpha diversity of the soil microbial
community
The rarefaction curves (Figure S3) of all samples tended to be
flat, suggesting that each sample was equally and reasonably
sampled. The coverage was over 0.99 in all samples, reveal-
ing that the sequencing depth was enough to capture almost
all the diverse bacterial and fungal communities in this study
(Table 2). The richness and diversity of bacterial and fungal
communities were different under various fertilization treat-
ments. The richness of bacteria and fungi (Chao1 and ACE)
were both NK > NPK > NP > PK fertilization, and there were
significant differences between treatments. The diversity of
soil bacteria and fungi (Simpson and Shannon) was ranked as
NK, NPK > NP > PK fertilization and the differences were
significant. Therefore, compared with the NPK treatment, NK
treatment increased the soil microbial diversity, while the PK
and NP treatments did the opposite impact.
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212 LI ET AL.

DIAGRAMA DE CAJAS Y BIGOTES

Valor máximo

Valor mínimo

Mediana

Media

Cuartil Q3

Cuartil Q1

Rango intercuartil

Valor atípico (outliers)

F I G U R E 1 Variation and distribution of grain yields for (a,b) rice and (c,d) rapeseed under different fertilization treatments PK, NK, NP, and
NPK from 2011 to 2020. The box plots in (b) and (d) display the distribution of rice and rapeseed yields, respectively, over the 10 years at different
treatments. A dot mark in the box indicates the mean value of crop yield. The upper, middle, and lower limits of each box denote, respectively, the
75th, 50th, and 25th percentages for crop yield, while different lowercase letters indicate significant differences in mean grain yield between
treatments at p < 0.05 in (b, d). *, **, *** indicate significant differences at p < 0.05, p < 0.01, p < 0.001 levels, respectively

TA B L E 1 Effects of long-term fertilization on soil chemical properties in bulk soils

Available N Olsen-P Available K Slowly available K

Treatment pH SOM (g−1 kg−1 ) (mg−1 kg−1 ) (mg−1 kg−1 ) (mg−1 kg−1 ) (mg−1 kg−1 )
PK 6.49 ± 0.28 a 29.5 ± 1.1 c 241.3 ± 28.5 c 4.23 ± 0.16 a 92.9 ± 6.3 b 520.6 ± 14.2 ab
NK 5.86 ± 0.17 b 31.1 ± 1.1 ab 263.2 ± 18.3 b 3.75 ± 0.90 b 98.0 ± 9.0 c 524.4 ± 15.4 a
NP 5.72 ± 0.13 bc 30.4 ± 0.9 b 259.7 ± 13.0 b 4.52 ± 0.23 a 72.3 ± 7.1 d 502.3 ± 9.7 b
NPK 5.68 ± 0.16 c 33.5 ± 1.2 a 286.2 ± 14.1 a 4.36 ± 0.38 a 115.8 ± 2.9 a 535.6 ± 34.2 a
Initial value 6.40 ± 0.19 a 28.8 ± 0.8 c 275.3 ± 10.7 ab 4.44 ± 0.49 a 96.6 ± 7.2 b 528.2 ± 21.5 a

Note: Means of three replicates are presented with standard deviation. Different letters in the same column indicate significant differences among four treatments and
initial values at the p < 0.05 level.
Abbreviation: SOM, soil organic material.

3.4 Composition of the soil microbial
community
Long-term fertilization altered the relative abundance of bac-
terial and fungal phyla in soil. Of the top 20 bacterial phyla
(Figure 2a), Proteobacteria and Acidobacteria had the highest
relative abundance in each treatment, belonging to the pre-
dominant bacterial community (relative abundance > 10.0%),
with an average of 31.9% and 14.2%, respectively. The rela-
tive abundances of Actinobacteria, Chloroflexi, Nitrospirae,
 14350645, 2023, 1, Downloaded from https://acsess.onlinelibrary.wiley.com/doi/10.1002/agj2.21264 by Cochrane Costa Rica, Wiley Online Library on [19/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
LI ET AL. 213

TA B L E 2 Richness and diversity of bacterial and fungal gene sequences in the soil samples

Richness index Diversity index

Treatment Chao1 ACE Simpson Shannon Coverage
Bacteria
PK 2225 ± 134 d 2138 ± 128 d 0.983 ± 0.059 a 8.62 ± 0.05 c 0.996 ± 0.052 a
NK 4215 ± 158 a 4187 ± 157 a 0.998 ± 0.030 a 10.78 ± 0.03 a 0.996 ± 0.031 a
NP 2894 ± 150 c 2822 ± 142 c 0.992 ± 0.052 a 9.31 ± 0.04 b 0.995 ± 0.052 a
NPK 3622 ± 172 b 3544 ± 163 b 0.997 ± 0.047 a 10.22 ± 0.04 a 0.994 ± 0.044 a
Fungi
PK 147 ± 9 d 138 ± 8 d 0.898 ± 0.038 b 4.95 ± 0.03 c 0.999 ± 0.042 a
NK 662 ± 25 a 645 ± 25 a 0.974 ± 0.037 a 6.68 ± 0.03 a 0.999 ± 0.038 a
NP 239 ± 11 c 223 ± 10 c 0.964 ± 0.036 a 5.91 ± 0.04 b 0.999 ± 0.045 a
NPK 553 ± 20 b 536 ± 28 b 0.978 ± 0.052 a 6.95 ± 0.04 a 0.999 ± 0.053 a

Note: Means of three replicates are presented with standard deviation. Different letters within one column indicate significant differences among four treatments at the
p < 0.05 level.
Abbreviation: ACE, abundance-based coverage estimator.

F I G U R E 2 The relative abundance of top 20 taxonomic groups for (a) bacteria and (b) fungi at the phylum level between different treatments
in 2020 and significant changes in (c) bacterial and (d) fungal key phylotypes under nutrient deficiencies identified using linear discrimination
analysis (LDA) linear discriminant analysis effect size

214
Latescibacteria, and Rokubacteria were higher, with aver-
age of 6.1%, 2.3%, 1.7%, 1.4%, and 1.1%, respectively. In
the fungi phylum, Ascomycota was the dominant species,
with an average relative abundance of 49.7%, followed by
Basidiomycota and Mortierellomycota with an average rela-
tive abundance of 10.4% and 4.4%, respectively. Compared
with the NPK fertilization, Proteobacteria, Actinobacte-
ria, Chloroflexi, Verrucomicrobia, and Planctomycetes in
the PK, NK, and NP treatments all increased significantly,
with the increase rates being 8.1%–14.6%, 23.5%–50.9%,
18.4%–50.8%, 93.9%–414.0%, and 122.6%–396.4%, respec-
tively (p < 0.05); however, Acidobacteria declined to varying
degrees by 29.3%–51.4%. The relative abundances of Bac-
teroidetes, Gemmatimonadetes, and Firmicutes were different
in the NK, PK, and NP treatments. Compared with the NPK
fertilization, Bacteroidetes, Gemmatimonadetes, and Firmi-
cutes in the NK fertilization significantly increased (p < 0.05)
by 55.7%, 14.4%, and 43.3%, respectively. At the same time,
Bacteroidetes, Gemmatimonadetes, and Firmicutes in the PK
and NP treatments decreased at an average of 57.9%, 88.8%,
and 34.6%, respectively.
As for the fungal community (Figure 2b), compared with
the NPK treatment, the relative abundance of Ascomycota
significantly reduced by 17.5% (p = 0.023), and the relative
abundance of Basidiomycota and Mortierellomycota signif-
icantly increased by 175.3% and 69.0%, respectively, in the
NK treatment. The relative abundances of Basidiomycota,
Mortierellomycota, and Rozellomycota in the NP treatment
were reduced by 17.7%, 45.6%, and 77.2%, respectively
(p < 0.05), compared with those of the NPK treatment. In
comparison, Olpadiomycota and Glomeromycota in the PK
treatment increased by 287.3% and 2428.6%, respectively.
At the level of bacterial phylum, Proteobacteria, Rokubac-
teria, and Planctomycetes were found to be the significantly
altered taxa in the PK fertilization (Figure 2c). Actinobacteria,
Gemmatimonadetes, Bacteroidetes, Chloroflexi, Verrucomi-
crobia, and Firmicutes were found to be sensitive to the
NK fertilization. Interestingly, the predominant Nitrospi-
rae, Latescibacteria, and Patescibacteria were significantly
altered in the NP fertilization, and only Acidobacteria
was positively altered in the recommended NPK fertilizer
application. At the level of fungal phylum (Figure 2d),
Glomeromycota, Olpidiomycota, and Rozellomycota were the
significantly altered taxa in the PK treatment. Basidiomycota,
Mortierellomycota, Zoopagomycota, and Chytridiomycota
were sensitive to the NK treatment. Conversely, Ascomycota,
Entorrhizomycota, and Kickxellomycota clearly changed in
the NP treatment. Mucoromycota and Aphelidiomycota were
altered in noticeable manner in the NPK treatment.
At the bacterial genus level (Figure 3a), Anaeromyxobacter
was the dominant genus, with an average relative abundance
of 6.27%, followed by Geobacter, Haliangium, Candidatus
Solibacter, and Nitrospira, with an average relative abundance
of 0.74%, 0.73%, 0.65%, and 0.51%, respectively. Among
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LI ET AL.
them, Anaeromyxobacter had the highest relative abundance
in the PK and NP treatments, and Haliangium and Candida-
tus Solibacter (p < 0.05) had the highest relative abundance
in the NK fertilizer application.
At the genus level of fungi (Figure 3b), the dominant
species were Fusarium and Mortierella, having an average rel-
ative abundance of 5.4% and 4.4%, respectively, followed by
Xylodon, Lecanicillium, Umbilicaria, and Chaetomium, with
an average of 2.7%, 2.4%, 2.3%, and 2.0%. The relative abun-
dance of Fusarium (p < 0.05) decreased significantly in the
PK and NP treatments than in the NPK treatment. Among all
treatments, Mortierella and Xylodon in the NK treatment had
the highest relative abundance, while Lecanicillium, Umbili-
caria, and Chaetomium (p < 0.05) had the lowest relative
abundance.
3.5 Beta diversity of the soil microbial
community
The Venn diagram (Figures 4) shows that the microbial pop-
ulation had both shared components and unique parts. The
PK, NK, and NP treatments shared 3.92%, 13.76%, and 7.04%
of the bacterial OTUs with the NPK treatment, while the
unique OTUs of the PK, NK, NP, and NPK treatments were
12.36%, 28.16%, 15.81%, and 20.69%, respectively. The PK,
NK, and NP treatments shared 3.38%, 13.19%, and 5.73%
of fungal OTUs with the NPK treatment, while the unique
OTUs of the PK, NK, NP, and NPK treatments were 6.67%,
36.5%, 10.52%, and 27.63%, respectively. Additionally, prin-
cipal coordinate analysis showed that bacterial communities
in the NK and NPK treatments were distinct from those in
the PK and NP treatments along the x-axis (Figure 4c), and
the first principal component (x-axis) accounted for 72.2% of
the total variation. Similarly, the first two principal coordi-
nates represented 79.0% of the variation in fungi (Figure 4d)
communities, in which the first principal component (x-axis)
accounted for 50.6% of the total variation.
To further compare the differences in the microbial com-
positions of the four treatments, a heatmap was used to
demonstrate the genus composition. The distribution of domi-
nant bacteria in each treatment was well marked; specifically,
in the NK treatment, up to 16 species could be identified in
the number of abundant bacteria (Figure 5). On the contrary,
the abundant bacteria in the PK, NP, and NPK treatments
were 10, nine, and five types of species, respectively. Further-
more, the cluster analysis results reflected that the PK and NP
treatments were similar, and so were the NK and NPK treat-
ments (Figure 5a). The results of the fungal genus also showed
that the distribution and relative abundance of the dominant
fungal groups in each treatment were significantly different
(Figure 5b). At the same time, the cluster analysis also indi-
cated that the NK and NPK treatments were similar and the
PK and NP treatments were homogeneous.
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LI ET AL. 215

F I G U R E 3 The relative abundance of preponderant taxonomic groups for (a) bacteria and (b) fungi at the genus level between different
treatments in 2020. The data represent the mean values of the three replications

F I G U R E 4 Venn graph of (a) bacterial and (b) fungal operational taxonomic units from soil samples and the principal coordinate analysis
(PCoA) plot of the dissimilarity between the treatments for (c) bacteria and (d) fungi based on Bray–Curtis differences in 2020

3.6 Correlation of dominant microbial
communities with soil properties
The dominant microbial groups of four treatments were
separated from each other in the two-dimensional sorting
(Figure 6), indicating that long-term unbalanced fertilization
had caused the differentiation of the soil microbial commu-
nity (bacteria p = 0.002 and fungi p = 0.002). The available
K, pH, slowly available K, SOM, and Olsen-P had a notewor-
thy impact on the bacterial community, which explained the
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216 LI ET AL.

F I G U R E 5 The heatmap displays the dominant (a) bacteria and (b) fungi variances among the treatments in 2020 at the genus level. The red
color represents genera with higher relative abundance in the corresponding sample, and the green color represents genera with lower relative
abundance

F I G U R E 6 Redundancy analysis (RDA) demonstrate the relationships between soil property indexes (red arrow) and the dominant (a)
bacterial and (b) fungal community (blue arrow) among soil samples. The direction of arrows indicates correlation with the first two canonical axes
and the length of arrows represents the strength of the correlations. SOM, soil organic matter

variation by 53.5%, 23.1%, 11.6%, 6.9%, and 4.8%, respec- 3.7 Potential pathways influencing crop
tively. Also, the Olsen-P, slowly available K, pH, available K, yield
and SOM had a prominent impact on the fungal community,
and the variations were explained by 61.2%, 17.7%, 9.2%, The results showed that the final model had a GOF of 0.73
8.6%, and 3.2%, respectively. and 0.56 for the bacterial and fungal groups, respectively
 14350645, 2023, 1, Downloaded from https://acsess.onlinelibrary.wiley.com/doi/10.1002/agj2.21264 by Cochrane Costa Rica, Wiley Online Library on [19/02/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
LI ET AL. 217

F I G U R E 7 Partial least squares path models for the rice and rapeseed yield in 2020. (a,b) Arrows indicate causal relationships, supplemented
by a path coefficient, and continuous and dash lines indicate positive and negative relationships. (c,d) The amount of the variability is explained by
the variables. Path coefficients are calculated after 1000 bootstraps. BC1, Bray–Curtis PCoA1; BC2, Bray–Curtis PCoA2; GOF, goodness of
fit; SOM, soil organic matter; UP1, unweighted UniFrac PCoA1; UP2, unweighted UniFrac PCoA2; WP1, weighted UniFrac PCoA1; WP2,
weighted UniFrac PCoA2

(Figure 7). The pathways of soil properties, alpha diver-
sity, and bacterial community composition together explained
72.8% of the total variance in rice and rapeseed yield, whereas
those three items represented 41.3% of the variation in the
fungal group. This suggested that soil proprietary (path coeffi-
cient = 0.79) had a greater impact on crop yield than microbial
properties, especially the bacterial group had more influence
than the fungal group on crop yield.
4 DISCUSSION
4.1 Effects of long-term fertilization on
rotation crop yields
Reasonable application of mineral fertilizers is necessary
for enhancing crop production and sustainable soil fertility.
Related studies have shown that the individual contribution
rate of NPK fertilizers on the yield of rice and rapeseed
was 20.3%, 12.2%, and 8.2% and 41.9%, 21.4%, and 11.5%,
respectively (Bai et al., 2015; Li et al., 2017; Yan et al., 2019).
Therefore, the nutrient limiting factor sequence of rice field
is generally manifested as N > P > K and rapeseed is more
sensitive to nutrient deficiency stress than rice in the same
site, which is consistent with the contribution rate of fertilizer
(Chen et al., 2020). However, our results showed P > N > K
fertilizer (Figure 1), which may be related to the lower avail-
able P content in this region compared to available N and
available K content (Table 1). However, compared to rice
season, soil moisture and temperature conditions reduce the
lower P bioactivity in the rapeseed season (Li & Zhong, 2021).
From the dynamic changes of crop yield in 10 years, the
decrease rate of the NP treatment was slightly better than that
of the NK and PK treatments in the paddy-upland rotation
compared to that of the NPK treatment, and the drop ten-
dency was noticeable in the rapeseed season. Compared with
the initial soil properties, the available K content in the end of
experiment was higher than the content of available N and
available P. With the extension of planting years, the sup-
ply capacity of soil fertilizer gradually decreased, which will
lead to a reduction in crop yield (Tang et al., 2018; Yousaf
et al., 2017). Moreover, the results shown in Figure 7 indi-
cate that the soil properties had more direct effect than the
community composition and the alpha diversity of bacterial

218
and fungal groups on crop yield. Therefore, nutrient uptake
by rice mainly comes from the soil stores, whereas rapeseed
depends more on exogenous NPK fertilizers (Li et al., 2017;
Zhang et al., 2020).
4.2 Effects of long-term fertilization on soil
chemical properties
The results demonstrated that after 10 years of fertilization,
the initial values of the physiochemical properties of soil
underwent a significant change (Table 1). These findings once
again illustrated the importance of balanced fertilizer applica-
tion to China’s food security and soil health. Compared to the
PK treatment, the pH values in the NK, NP, and NPK treat-
ments had a varying amount of reduction, especially the NPK
treatment. Previous research had shown that the application
of large amounts of N fertilizer could cause soil acidification.
There are two factors involved the soil acidification: (1) the
exchange of NH4 + -N with H+ on root surface and (2) the nitri-
fication of unutilized NH4 + -N in soil, which will produce H+
and lower the soil pH and microbial population (Yan et al.,
2020). There, the combination of organic and inorganic fertil-
izers can reduce soil acidification, and thus improve microbial
diversity.
4.3 Effects of long-term fertilization on soil
microbial alpha diversity
Alpha diversity of soil bacterial communities plays an impor-
tant role in the sustainable development of agriculture (Eo &
Park, 2016; Guo et al., 2020). The NK treatment had the high-
est richness and diversity index, and the PK treatment had the
lowest values, which was consistent with the findings of Ling
et al. (2017). However, the results of Islam et al. (2012) indi-
cated that the addition of NPK fertilizer had little effect on
microbial activity with long-term fertilization. This contradic-
tion may be related to the level of soil fertility and the duration
of the field experiment. From the fungal alpha diversity, the
change tendency in different treatments was like that of bac-
teria. Fungi occupies a large proportion of the soil microbial
community, and there are many fungi distributed in the soil
environment. They can decompose organic matter and can
be used as a significant biological indicator to evaluate the
soil quality. For crops, they have an effective nutrient supply
function; for example, the arbuscular mycorrhizal fungi could
directly activate mineral-N and K and transfer to host plant
(Miransari, 2011). As shown in Table 1, the available P limi-
tation in our experiment was remarkable, which caused great
changes in the composition of P-related microbial species.
Bulgarelli et al. (2019) found that the alpha diversity index of
the fungal community was significantly decreased after con-
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LI ET AL.
tinuous application of P fertilizer in eucalypts forest. There
were also studies suggesting that long-term application of P
fertilizer increased the diversity of soil bacteria (Ling et al.,
2017; Wasaki et al., 2018).
The diversity of bacteria and fungi in the PK treatment
was the lowest (Table 2), indicating that the soil available N
content significantly affects the vitality of microorganisms.
Therefore, for soil microorganisms, soil N availability is still
the primary limiting factor (Miransari, 2011; Zeng et al.,
2016). The correlation analysis of soil properties and diver-
sity index (Figure S4) implied that the Simpson and Shannon
indexes of bacteria and fungi were significantly positively cor-
related with SOM and available N and that they had a negative
correlation with pH and Olsen-P. Previous studies showed that
the adverse impact of soil acidification in paddy soil caused
by the superfluous application of N or NPK fertilizer on soil
microbial diversity far exceeded the positive effect of fertiliza-
tion (Ullah et al., 2020). Therefore, applying organic fertilizer
in combination with NPK chemical fertilizer was reported
more effective than providing NPK fertilizer alone in future
(Liu, Chen, et al., 2020; Wang et al., 2017).
4.4 Effect of long-term fertilization on the
composition of soil microbial community
The predominant bacterial phyla (Figure 2) were Acidobacte-
ria, Proteobacteria, and Actinobacteria, and this is consistent
with the phyla reported by Wang et al. (2018) and Guo et al.
(2019). Proteobacteria, Nitrospirae, Firmicute, and Acti-
nobacteria (i.e., R-strategist) are considered as copiotrophic
groups, while Acidobacteria, Actinobacteria, Chloroflexi, and
Planctomycetes were typical oligotrophic (i.e., K-strategist)
bacteria (Fierer et al., 2012). Sun et al. (2015) found that
the relative abundance of Proteobacteria was significantly
positively correlated with soil carbon and N content, which
is consistent with our results (Figure 6). Regarding the
discrepancy, the relative abundances of Bacteroidetes, Gem-
matimonadetes, and Firmicutes without P fertilization (NK
treatment) increased markedly, while Bacteroidetes, Gemma-
timonadetes, and Firmicutes without N and K fertilization
decreased significantly. These results showed that different
nutrient fertilizer led to major changes in the relative abun-
dance of particular bacterial communities (Miransari, 2011).
Both long-term and short-term studies have found that the
abundance of Acidobacteria decreased significantly with the
increase of N fertilizer application, which was closely asso-
ciated with soil pH value (Liu, Li, et al., 2020), while, in
our study, the relative abundance of Acidobacteria, serving
as typically oligotrophic bacteria in the recommended NPK
fertilization, was higher than that in other fertilization, which
was in contradiction with previous research (Eo & Park, 2016;
Islam et al., 2012). This may be related to the increase in the

LI ET AL.
special subgroup function of Acidobacteria, which, in terms
of genus, were unclassified Subgroup 3, unclassified Sub-
group 6, unclassified Subgroup 17, and uncultured Desul-
fovirga sp. (Figure 5). Additionally, balanced fertilization
could stimulate the propagation of Acidobacteria phylum
community, thereby activating the insoluble mineral ions in
the soil (Sun et al., 2015; Wang et al., 2017).
In the fungi groups, Basidiomycota (oligotrophic) and
Ascomycota (eutrophic) were the dominant phyla communi-
ties. Figure 2 shows that the NK fertilization significantly
reduced the abundance of Ascomycota and increased the rel-
ative abundance of Basidiomycota and Mortierellomycota,
which is in accordance with the findings of Wang et al. (2018).
The relative abundances of Basidiomycota, Mortierellomy-
cota, and Rozellomycota in the NP fertilization decreased
obviously compared with the NPK fertilization. In compari-
son, Olpadiomycota and Glomeromycota in the PK treatment
increased by varying degrees (Figure 2). This again proves
that the unbalanced fertilizer input leads to an increase in olig-
otrophic fungi and a decrease in eutrophic fungi. Particularly,
according to the results of the cluster analysis at the genus
level, there were significant differences in the distribution of
fungal species among the four treatments (Figure 3), which
may be closely related to soil nutrient status and response to
crop yield. Fan et al. (2020) found that the microbial resistance
indirectly drives the effects of nutrient fertilization on plant
production. This phenomenon was also found in the results of
RDA, that is, Olsen-P and slowly available K had the positive
influence on the fungal community and rice or rapeseed yield
(Figures 6 and 7). The reasons the microbe influences the
crop production may be that (1) balanced fertilization could
facilitate plants acquiring more nutrients and less competi-
tion from microbial species in soil and (2) abundant nutrient
could higher benefit bacteria and lower relative abundance of
potential fungal plant pathogens (Fan et al., 2020; Li & Zhong,
2021). Our study also indicated that soil properties altered
by fertilization management had a more direct effect on crop
yield than the community composition and the alpha diversity
of bacterial and fungal groups. Therefore, the balanced fertil-
ization with currently recommended NPK fertilization could
enhance the paddy-upland rotation production as well as the
soil microbial population and diversity.
5 CONCLUSIONS
Long-term deprivation of fertilizer input significantly influ-
enced paddy-upland rotation yield and soil properties, espe-
cially pH and available K content. Compared with NPK
fertilization, non-N, -P, and -K fertilization led a decline in
the average annual yield of rice and rapeseed. Rapeseed was
more sensitive to insufficient fertilizer input than rice in the
same site in the rotation system. The NK treatment increased
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219
the soil microbial diversity, whereas the PK and NP treatments
had a contrary impact, compared with the NPK treatment. The
unbalanced fertilization treatment increased the abundance of
oligotrophic bacteria, such as Actinobacteria, Chloroflexi, and
Planctomycetes, and the relative abundance of Basidiomy-
cota and Mortierellomycota. The application of NPK fertilizer
increased the abundance of the copiotrophic bacteria, such
as the Firmicutes, Gemmatimonadetes, and Proteobacteria
phyla, and the relative abundance of Ascomycota. Available
K, pH, and slowly available K were closely related to alter-
ations in the composition of the soil bacterial community,
while, Olsen-P, pH, and slowly available K were significantly
correlated with the fungal community. Soil properties altered
by fertilization management had a more direct effect on crop
yield than the community composition and the alpha diversity
of bacterial and fungal groups.
AU T H O R C O N T R I B U T I O N S
Jifu Li: Project administration; writing – original draft;
writing – review & editing. Guoyu Gan: Data curation;
investigation. Qianqian Zeng: Investigation.
AC K N OW L E D G M E N T S
This work was supported in part by the Open-end Fund
of Engineering Research Center of Ecology and Agricul-
tural Use of Wetland, Ministry of Education (KF2020015)
and the Open Fund of Key Laboratory of Fertilizer Uti-
lization, Ministry of Agriculture and Rural Affairs, China
(KLFAW201901). The authors earnestly thank the editors and
anonymous reviewers for their work.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Jifu Li https://orcid.org/0000-0002-3639-9434
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S U P P O RT I N G I N F O R M AT I O N
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
How to cite this article: Li, J., Gan, G., & Zeng, Q.
(2023). Impacts of long-term fertilization on crop
yield and microbial communities under rice–rapeseed
rotation. Agronomy Journal, 115, 208–221.
https://doi.org/10.1002/agj2.21264